Cell-free DNA (cfDNA) exists in a variety of body fluids in the human body, is an important research object for the early screening, diagnosis, monitoring and prognosis of many diseases, especially cancer, and has great potential as a non-invasive biomarker. The mechanisms of cfDNA production are complex and diverse, and apoptosis, necrosis and active cell secretion are recognized as the main sources. The cfDNA fragments produced by apoptosis and cell secretion are smaller, while the cfDNA fragments produced by cell necrosis are larger. According to the difference in fragment size, cfDNA is divided into small molecular weight fragments less than 1 kb and large molecular weight fragments more than 1 kb, and fragments higher than 10 kb are considered to be genome DNA with high integrity (gDNA). 90% of cfDNA exists in low molecular weight and most fragments are 80-200 bp in size.
Compared to the extensive study of cfDNA in blood, the current study of cfDNA in urine is not comprehensive. Urine cfDNA not only contains free nucleic acids filtered through the glomeruli from the blood circulation system, but also nucleic acid fragments directly released into the urine by the urinary system and the apoptosis and necrosis of urothelial cells. The wide range of sources makes urine cfDNA more diverse in size and morphology. In the daily work of the biobank of Zhongnan Hospital of Wuhan University, urine samples from patients with urinary diseases (mainly bladder tumors) were selected for cfDNA extraction and storage. The concentration, purity, fragment size and function of urine cfDNA were detected and analyzed in the sample bank. The median concentration of cfDNA samples from 467 patients was 16.70 ng/μl and the maximum was 1710 ng/μl. Only 50% of the samples have high purity, and the A260/A280 value is between 1.70 and 2.00. For the samples with high purity, the concentration values measured by different instruments are more similar. The urine cfDNA fragments are mainly about 180 bp, and the gradient bands of about 180 bp are often seen. The distribution of large molecular weight nucleic acid fragments is about 1.5 kb and 8 kb. The high proportion of large fragments in urine cfDNA samples may be related to the necrosis of epithelial cells shed from kidney, bladder, prostate and other tissues of patients with urinary diseases. Our results further verified the possible production pathway of urine cfDNA.
Because there are more salts and metabolites in urine, cfDNA samples are prone to impurity contamination. When UV spectrophotometer is used to determine DNA concentration, impurity causes a large deviation of absorbance at 260 nm, and the concentration value is inaccurate. The principle of fluorometers and bioanalyzers is that specific fluorescent dyes bind to DNA and can measure DNA concentration more accurately. When the sample purity is high, the concentration values measured by the detection instruments of different principles are relatively close, on the contrary, the concentration values are different. According to the difference of sample concentration values measured by different instruments, the sample purity can be indirectly assessed.
At present, the quality assessment of cfDNA has not yet formed a unified standard, the existing detection methods are mainly divided into two categories: one is the non-specific sequence concentration, purity and fragment size detection, commonly used spectrophotometer, fluorometer, bioanalyzer and other instruments; The second is sequence specific detection, such as ordinary PCR, real-time quantitative PCR, digital PCR and high-throughput sequencing. The vast majority of cfDNA fragments in blood are small molecular weight and low concentration, while the concentration range of cfDNA in urine is large and there are many large fragments. A spectrophotometer with low sensitivity but wide concentration detection range can be selected for preliminary measurement of sample concentration and purity. Subsequently, 4200 TapeStation combined with gDNA strips or High Sensitivity D5000 strips were selected to measure more accurate concentrations and check the size of nucleic acid fragments.
